29  192J 


TAUB 

DISSERTATION. 


QP 

a. 


Reprinted  from  THE  AMERICAN  JOURNAL  OF  PHTSIOLOQT 
Vol.  69,  No.  1,  February,  1922 


-OGlf 

LIBRARY 
G 


STUDIES  IN  THE  REGENERATION  OF  BLOOD1 

ZALIA  JENCKS 

From   the   Sheffield   Laboratory   of  Physiological   Chemistry,    Yale    University, 

New  Haven,  Connecticut 

Received  for  publication  September  8,  1921 

Studies  in  the  regeneration  of  blood  after  simple  anemia  have  been 
made  on  many  species  of  animals  and  on  man.  Although  various  phases 
of  the  growth  of  blood  have  been  extensively  investigated  in  connection 
with  destruction  of  the  erythrocytes  by  chemical  agents  or  specific 
diseases,  much  less  information  is  available  regarding  recovery  from 
the  anemias  produced  by  hemorrhage. 

Many,  if  not  most,  of  the  earlier  researches  lack  a  convincing  char- 
acter because  relatively  few  of  the  variables  have  been  sufficiently 
taken  into  account.  Heretofore  the  work  on  blood  regeneration  has 
been  on  animals  whose  dietary  control  was  not  complete.  Inasmuch 
as  the  more  recent  investigations  of  nutrition,  conducted  on  laboratory 
animals,  have  made  it  possible  to  control  a  variety  of  dietary  factors, 
only  one  variable  will  need  to  be  taken  into  consideration  for  the  dura- 
tion of  a  particular  series  of  experiments. 

The  present  studies  on  albino  rats  were  undertaken  because  of  the 
relatively  large  amount  of  information  available  concerning  the  dietary 
requirements  of  these  animals.  The  rat  is  small  enough  to  make 
possible  the  use  of  large  numbers  for.  experimental  purposes  and  it  has 
been  employed  as  a  laboratory  animal  for  so  many  generations  that 
laboratory  conditions  do  not  make  its  confinement  unnatural.  Our 
rats  were  from  the  colony  of  the  Connecticut  Agricultural  Experiment 
Station.  They  were  kept  on  a  standard  synthetic  diet2  while  in  experi- 

1  Material  for  this  paper  was  taken  from  the  dissertation  submitted  for  the 
degree  of  Doctor  of  Philosophy,  Yale  University,  1921. 

3  Standard  diet  used :  per  <**t 

Cornstarch 50.5 

Casein 18.0 

Lard 18.0 

Washed  butter 9.0 

Salt  mixture 4.5 

240 


STUDIES   IN   THE   REGENERATION    OF   BLOOD  241 

mental  use  and  during  all  of  the  time  when  they  were  not  under  direct 
observation.  This  eliminated  complications  arising  from  having  the 
animals  eat  such  complex  mixtures  as  they  would,  had  they  been  fed 
table  scraps. 

Technique.  The  preliminary  experiments  were  designed  to  discover 
the  limit  of  successful  bleedings  in  the  rat,  the  period  for  regeneration 
of  blood  after  hemorrhages,  and  to  find  the  effect  of  the  dietary  factors 
on  such  regeneration. 

The  rats  on  which  hemorrhages  were  produced  were  kept  in  iron-wire 
cages  with  paraffined  tin  cups  for  water  and  porcelain  cups  for  food. 
The  methods  and  technique  employed  have  been  described  by  Miss 
Ferry  (1).  The  standard  synthetic  diet  developed  by  the  work  of 
Osborne  and  Mendel  (2),  (3),  (4)  and  others  was  used  as  a  basis  for 
all  the  dietaries  employed.  Adult  and  young  animals  of  both  sexes 
were  used,  varying  in  average  weight  from  100  grams  for  the  young 
to  300  grams  for  the  adults.  The  tables  of  Donaldson  (5)  were  carefully 
followed  in  estimating  the  blood  volume  of  each  experimental  animal 
used. 

Experimental  technique.  The  blood  of  every  rat  was  carefully  ex- 
amined for  the  number  of  erythrocytes  and  percentage  of  hemoglobin 
before  any  hemorrhages  were  made.  The  counts  were  checked  in  every 
case  during  the  first  2  months  until  the  operator  had  attained  sufficient 
skill  to  make  such  duplications  unnecessary.  The  method  of  blood 
count  was  the  familiar  one  with  a  Thoma-Zeiss  hemocytometer  and  the 
use  of  either  Hayem's  or  Toisson's  solution  for  blood  dilution.  Hemo- 
globin was  determined  by  the  colorimetric  procedure  of  Cohen  and 
Smith  (6) .  Rat  blood  was  used  as  a  standard  and  its  color  was  checked 
by  fresh  material  at  frequent  intervals. 

The  sample  of  blood  for  examination  was  taken  from  a  drop  extruded 
when  the  end  of  the  tail  was  snipped.  The  rat  was  placed  in  an  open 
glass  bottle,  the  bottom  of  which  had  been  removed  and  a  tin  cap  with 
a  hole  for  the  tail  placed  over  the  opening;  leather  straps  from  the  neck 
of  the  bottle  held  the  rat  securely  in  this  armor  (see  fig.  1).  This 
arrangement  allowed  free  manipulation  of  the  tail  by  the  experimenter. 
The  skin  of  the  tail  was  softened  with  mineral  oil.  Previous  to  removing 
the  end  of  the  rat's  tail  it  was  placed  in  warm  water  or  brushed  over 
with  toluene  in  order  to  suffuse  the  blood  vessels.  There  are  four  blood 
vessels  in  the  rat's  tail,  one  dorsal,  one  ventral  and  two  lateral.  The 
blood  was  obtained  from  these  by  manipulating  the  tail  after  the  fashion 
of  milking  and  allowing  the  fluid  thus  "milked"  out  to  drop  into  a 


242 


ZALIA   JENCKS 


tared  glass  dish.  At  the  beginning  of  this  series  of  experiments,  hemor- 
rhages of  from  one-third  to  one-half  of  the  estimated  total  blood  volume 
were  produced.  After  "milking  out"  20  or  30  drops  a  clot  usually 
formed ;  this  was  carefully  wiped  off  with  a  previously  weighed  piece 
of  cotton  and  added  to  the  blood  in  the  dish.  Then  more  blood  was 
worked  out  of  the  end  of  the  tail.  The  blood  and  vessel  were  weighed 
rapidly  to  centigrams — two  or  three  times,  if  necessary — until  at  least 
one-third  of  the  whole  blood  volume  contained  in  the  rat  had  been 
removed.  This  method  which  was  laborious  and  unsatisfactory  re- 
sulted in  occasional  injury  to  the  tail  accompanied  in  some  cases  by 


Fig.  1.    This  shows  a  rat  held  in  the  bottle  so  that  the  tail  is  exposed  for 
"milking"  out  blood. 

infection.  Frequently  a  second  hemorrhage  was  desired  before  the 
wound  on  the  tail  was  completely  healed.  Because  of  this  soreness 
and  the  possibility  of  infection  it  often  became  impossible  to  obtain 
a  sample  of  healthy  blood  for  cell  count  and  hemoglobin  determination. 
Subsequently,  therefore,  the  method  used  for  obtaining  blood  from 
guinea  pigs  in  serum  work  was  adopted.3  In  this  procedure  blood  is 
secured  directly  from  the  jugular  vein.  The  rat  was  anesthetized  with 
ether,  the  fur  closely  clipped  from  its  neck,  a  slit  about  \  inch  long  made 
in  the  skin,  the  jugular  exposed,  a  v-shaped  nick  made  in  the  vein  with 

3  Thanks  are  due  my  co-worker,  Dr.  W.  G.  Karr,  for  his  kindly  suggestion  as 
to  use  of  this  method  and  for  his  assistance  in  the  first  operations. 


STUDIES   IN   THE   REGENERATION   OF   BLOOD  243 

scissors,  and  the  animal  held  over  a  weighed  glass  vessel  into  which  the 
blood  was  allowed  to  drop.  If  care  was  exercised  it  trickled  slowly  and 
in  a  well-directed  stream  and  scarcely  touched  the  fur.  The  blood  was 
allowed  to  flow  into  the  dish  up  to  the  mark  previously  placed  to  indicate 
one-third  the  estimated  total  volume  of  blood.  By  the  time  this  had 
run  out  a  clot  had  usually  formed.  The  clot  was  carefully  and  gently 
wiped  off  from  the  wound  with  a  previously  weighed  bit  of  cotton,  and 
this  added  to  the  blood  in  the  dish,  and  the  whole  was  then  weighed. 
If  a  clot  failed  to  form  the  bleeding  was  stopped  by  pressing  a  small 
(weighed)  piece  of  cotton  against  the  cut  in  the  Vein.  Ordinarily  this 
proved  effective.  If  less  than  one-third  of  the  (estimated)  rat's  blood 
had  run  out  by  the  time  a  clot  had  formed,  it  was  easy  to  start  the 
bleeding  again  by  harshly  wiping  the  nick  in  the  vein  with  a  weighed 
pledget  of  cotton;  then  more  blood  dripped  and  a  second  weighing 
was  made.  This  second  bleeding  was,  however,  seldom  necessary. 
The  wound  in  the  skin  was  then  washed  with  dilute  alcohol  (25  per 
cent)  and  sewed  up.  The  rats  recovered  readily,  the  wound  healing 
in  from  2  to  3  days  in  every  case. 

Blood  counts  and  hemoglobin  determinations  were  made  daily  until 
the  original  values  returned.  During  the  period  of  regeneration  frag- 
mentary red  cells  were  often  seen  in  the  blood,  but  as  regeneration 
proceeded  these  became  fewer  in  number  and  finally  disappeared  alto- 
gether. The  number  of  leucocytes  increased  the  first  day  or  two  after 
the  hemorrhage,  but  not  in  great  abundance.  As  shown  by  Lamson  (7) 
the  number  of  erythrocytes  as  well  as  the  percentage  of  hemoglobin 
varies  among  the  individuals,  but  the  drop  after  hemorrhage  in  each 
case  is  comparable. 

Criteria  of  regeneration.  The  return  of  the  number  of  red  blood  cor- 
puscles and  the  percentage  of  hemoglobin  to  the  values  noted  before  any 
loss  of  blood  had  been  sustained  by  the  rat  were  the  criteria  used  in 
judging  regeneration.  These  values  remained  approximately  constant 
for  two  successive  days  before  the  rat  was  considered  to  have  regenerated 
his  blood.  For  normal  animals  of  either  sex  fed  on  standard  diet 
(see  footnote  2)  with  the  addition  of  dried  brewery  yeast,  the  time  was 
from  6  to  9  days  (table  1).  This  has  been  corroborated  by  the  sub- 
sequent studies  of  Geiling  and  Green  (8)  in  this  laboratory.  At  the 
end  of  the  period  required  for  regeneration,  in  many  cases  a  second 
hemorrhage  required  about  the  same  time  for  a  return  to  normal  blood 
conditions.  A  third  hemorrhage  "was  made  on  some  of  these  rats,  but 
regeneration  in  such  animals  always  needed  more  time.  If,  however, 


244 


ZALIA   JENCKS 


a  week's  time  intervened  between  return  to  normal  and  the  following 
bleeding,  the  regeneration  rate  was  normal.  Similar  experiences  have 
been  recorded  by  Geiling  and  Green  for  double  hemorrhages. 

Blood  regeneration  in  growing  rats.  To  study  the  effect  of  loss  of 
blood  on  the  growth  of  young  animals  bleedings  were  made  on  healthy 
growing  rats  fed  on  the  standard  diet  and  brewery  yeast  as  the  adults 
were  fed,  the  hemorrhage  procedure  being  the  same  as  for  the  grown 
rats.  The  time  of  total  regeneration  for  these  little  rats  was  from  7  to 
9  days  (see  table  2).  After  the  hemorrhage  they  recovered  quickly, 

TABLE  i 
Blood  regeneration  in  adult  rats  after  a  single  hemorrhage  on  the  standard  diet 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES* 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

220 

130 

5.5 

9,  360,  000 

91 

10,  136,  000 

104 

7 

210 

12.5 

5.5 

9,472,000 

100 

9,  168,  000 

87 

9 

272 

15.7 

5.5 

8,256,000 

94 

9,  008,  000 

87 

8 

310 

17.5 

7.0 

9,264,000 

104 

9,  504,  000 

91 

6 

250 

14.5 

5.1 

9,500,000 

110 

9,  104,  000 

98 

7 

210 

12.4 

5.0 

9,000,000 

94 

9,  100,  000 

96 

7 

300 

17.4 

5.0 

9,664,000 

107 

8,  800,  000 

91 

8 

*  The  figures  in  the  tables  under  the  heading  Red  Blood  Corpuscles  give  the 
number  of  ery  throcy  tes  before  hemorrhage  and  the  number  at  the  end  of  recovery. 

continued  to  grow  and  increase  their  body  weight  in  a  normal  manner. 
Apparently  the  loss  of  one-third  of  their  blood  did  not  hinder  growth 
in  any  observable  respect.  Some  young  animals  whose  growth  had 
previously  been  delayed  by  lack  of  vitamin  B  in  experiments  by 
Cajori  (9)  were  also  used,  but  these  recovered  in  the  same  way  as  the 
animals  showing  uninhibited  growth. 

Diet  and  dietary  factors.  In  order  to  make  these  experiments  as 
accurate  as  possible  and  to  eliminate  some  of  the  variables  occurring  in 
other  work  on  blood  regeneration,  the  standard  diet,  with  the  addition 
of  dried  brewery  yeast,  was  used.  Diets  of  each  component  of  this 


STUDIES   IN   THE   REGENERATION   OF   BLOOD 


245 


composite  food  were  used,  that  is,  protein  only  (casein) ;  carbohydrate 
only  (cornstarch) ;  fat  only  (washed  butter) ;  protein-free  food  (butter, 
lard,  cornstarch  and  salt  mixture)4  and  one  food  accessory  (vitamin 
B)  in  a  relatively  concentrated  form.  The  sources  of  the  food  accessory 

TABLE  2 
Blood  regeneration  in  growing  rats  after  a  single  hemorrhage  on  standard  diet 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

92 

6.1 

2.1 

11,264,000 

.      100 

8,500,000 

100 

9 

95 

6.3 

2.1 

9,  536,  000 

100 

7,  752,  000 

96 

9 

85 

6.0 

2.25 

10,880,000 

110 

9,312,000 

110 

9 

111 

7.2 

2.4 

8,  256,  000 

94 

7,442,000 

96 

7 

112 

7.2 

2.4 

10,  336,  000 

114 

9,440,000 

98 

7 

103 

6.77 

3.0 

9,504,000 

110 

8,  448,  000 

94 

7 

113 

7.2 

2.5 

10,  000,  000 

110 

9,  109,  000 

110 

7 

TABLE  3 


Blood  regeneration  in  adult  rats  after  a  single  hemorrhage  on  a  mixed  diet 


DIET 

BODY 
WEIGHT 

TOTAL 
BLOOD 

AMOUNT 
OF  HEM- 
ORRHAGE 

RED  BLOOD 
CORPULCLES 

HEMO- 
GLOBIN 

TIME  FOR 
REGEN- 
ERATION 

grams 

grams 

grams 

per  cmm. 

days 

322 

18.34 

8.2 

10,  192,  000 

94 

Dog  biscuit  

8,400,000 

98 

7 

320 

18.0 

6.3 

10,  080,  000 

100 

8,912,000 

98 

7 

Table  scraps  < 

310 

17.7 

5.25 

8,812,000 
8,  800,  000 

78 
96 

10 

were  dried  brewery  yeast,  yeast  concentrate5  (10),  tomato  juice  and 
orange  juice. 

4  Salt  mixture,  Osborne  and  Mendel,  Journ.  Biol.  Chem.,  1919,  xxxvii,  572. 
8  Yeast  concentrate,  Osborne  and  Wakeman  (10). 


246 


ZALIA   JENCKS 


Mixed  diets.  In  order  to  learn  what  difference,  if  any,  existed  between 
the  effect  of  our  standard  (synthetic)  diet  and  that  of  scrap  food  on  the 
rate  of  blood  regeneration,  two  rats  were  fed  on  dog  biscuit  for  2  days 
before  bleeding  and  then  during  the  entire  period  of  the  manufacture 
of  new  blood.  The  results  are  shown  in  table  3.  In  both  cases  the 
organism  regenerated  hemoglobin  more  rapidly  than  red  cells.  This 
may  have  been  merely  accidental,  but  the  same  circumstances  occurred 
in  the  case  of  one  rat  fed  on  table  scraps  (meat,  lettuce,  bread,  etc.). 
This  opens  the  question  as  to  what  factor  or  factors  are  here  present 
which  we  did  not  have  in  the  standard  basal  diet  with  added  yeast  as 
a  source  of  vitamin  B. 

TABLE  4 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage  on  a  limited  food  intake — 

5  grams  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOB 
REGENERA- 
TION 

grama 

grams 

grams 

per  cmm. 

days 

292 

16.7 

5.8 

10,  000,  000 

91 

9,  128,  000 

90 

7 

320 

18.3 

6.4 

8,  256,  000 

94 

8,  480,  000 

90     . 

7 

240 

14.0 

4.8 

9,360,000 

91 

'  8,  886,  000 

91 

7 

230 

13.4 

4.6 

9,  472,  000 

100 

9,  776,  000 

104 

6 

200 

11.9 

4.0 

7,  328,  000 

70 

8,  128,  000 

72 

6 

Underfeeding.  A  series  of  hemorrhages  was  made  on  animals  with 
a  limited  supply  of  food,  i.e.,  5  grams  per  animal  per  day,  including 
0.2  grams  yeast  daily  added  to  the  standard  basal  diet  as  above.  This 
limited  food  consumption  did  not  prolong  the  duration  of  regeneration 
appreciably,  the  normal  being  reached  from  6  to  7  days  after  the  initial 
loss  of  blood  (table  4) .  Loss  of  body  weight  occurred  in  each  case,  as 
might  be  expected  since  the  caloric  intake  was  insufficient,  but  the 
animals  remained  healthy  and  active  throughout  the  period  of  limited 
feeding. 

Starvation.  When  animals  were  not  allowed  any  food  throughout 
the  period  of  blood  regeneration,  the  rate  of  return  to  normal  hemoglobin 
value  and  number  of  erythrocytes  was  slower  than  in  the  above  cases, 
the  period  for  one  animal  being  as  long  as  13  days.  A  control  rat  with- 


STUDIES   IN   THE    REGENERATION    OF   BLOOD 


out  hemorrhage  was  starved  at  the  same  time;  its  blood  was  found  to 
vary  no  more  in  red  cell  count  and  hemoglobin  percentage  from  day  to 
day  than  that  of  a  normal  animal  eating  as  much  as  it  desired.  Normal 
rats  do  not  maintain  wholly  constant  values  for  either  hemoglobin 
percentage  or  number  of  red  cells.  This  slower  rate  of  regeneration 
during  inanition  is  in  agreement  with  the  results  of  Hooper  and 
Whipple  (11)  on  fasting  dogs  after  simple  anemia  (table  5). 

Protein  only.  Since  casein  is  the  protein  used  in  our  standard  synthetic 
diet  and  has  been  shown  to  be  an  adequate  protein  (2),  this  was  fed 
as  a  sole  source  of  nourishment  to  another  group  after  hemorrhage 
until  blood  regeneration  was  accomplished.  Only  2  grams  per  day  per 
animal  were  used  after  it  was  found  that  the  rats  refused  to  eat  more 
than  this  amount  in  that  period.  This  limited  protein  diet  was  used 
to  see  whether  in  accord  with  the  experience  gained  with  the  protein- 

TABLE  5 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  during  this  period  they 

received  no  food 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

340 

19.2 

7.5 

8,  860,  000 

114 

8,900,000 

110 

13 

330 

18.7 

6.6 

8,  562,  000 

74 

0,  728,  000 

87 

10 

rich  diet  of  dogs  (11)  the  rate  of  regeneration  was  more  rapid  than  on 
other  diets  of  one  food  factor  only. 

The  tune  of  blood  regeneration  in  this  set  of  experiments  was  from 
9  to  12  days,  more  rapid  than  in  starvation  but  not  so  rapid  as  on  some 
other  equally  limited  diets  (table  6).  It  must  be  borne  in  mind  how- 
ever that  no  food  accessories  (vitamins  A  and  B)  were  present  and 
hence  the  animals  ate  less  than  they  otherwise  would.  The  energy 
intake  was  obviously  inadequate  so  that  underfeeding  complicated 
the  problem.  In  all  these  feeding  periods  where  the  diets  were  limited 
in  amount  or  kind,  or  both,  the  rats  were  more  coprophagous  than 
normal,  no  doubt  in  an  effort  to  satisfy  their  desire  for  a  feeling  of 
fullness. 

Fat  only.  In  feeding  the  fat  portion  of  the  basal  diet,  butter  washed 
free  of  salt  was  used  in  an  amount  corresponding  to  the  daily  weight 
of  protein  fed.  The  rats  did  not  eat  the  butter  readily  and  never  ap- 


248 


ZALIA   JENCKS 


peared  so  anxious  for  their  food  as  did  the  animals  on  the  other  diets. 
The  time  required  for  regeneration  of  blood  by  this  set  of  fat-fed  animals 
was  9  days,  not  longer  than  for  the  protein-fed  rats  (table  7). 

Carbohydrate  only.  Two  grams  of  cornstarch,  made  in  the  form  of 
a  paste  with  water  to  prevent  scattering,  were  fed  daily  to  each  of  three 
rats  after  they  had  been  bled.  These  animals  required  12  days  for 

TABLE  6 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage  on  a  protein  diet — 

2  grams  casein  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OP 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

290 

16.7 

7.7 

10,656,000 

96 

10,  032,  000 

96 

9 

180 

11.9 

4.25 

11,  072,  000 

104 

10,  500,  000 

105 

12 

153 

10.2 

3.9 

10,432,000 

100 

9,600,000 

100 

12 

158 

10.56 

3.0 

10,336,000 

104 

10,  112,  000 

104 

9 

TABLE  7 


Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  diet  of  butter  fat  only 

— 2  grams  per  day 


BOOT  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

295 

17.0 

7.0 

8,528,000 

98 

9,  136,  000 

98 

9 

355 

20.0 

9.0 

8,480,000 

76 

7,800,000 

80 

9 

165 

11.0 

4.0 

10,416,000 

91 

9,440,000 

80 

9 

complete  regeneration.  This  is  somewhat  longer  than  for  those  on  the 
protein  diet,  in  agreement  with  the  work  on  dogs  receiving  a  carbohy- 
drate-rich diet  (11)  (table  8). 

Protein-free  food.  This  food  was  made  up  after  the  formula  for  the 
standard  diet  with  the  casein  replaced  by  corn  starch,  making  a  food 
containing  68.5  per  cent  corn  starch,  18  per  cent  lard,  9.0  per  cent 
butter  and  4.5  per  cent  salts.  Three  animals  were  given  5  grams  of 


STUDIES   IN   THE    REGENERATION   OF   BLOOD 


249 


this  protein-free  food  daily,  hemorrhages  were  made  and  the  process 
of  blood  regeneration  watched  by  the  methods  used  before.  These  rats 
replenished  their  blood  supply  in  from  9  to  12  days  (table  9),  slightly 
more  time  being  required  in  this  series  than  in  the  case  of  the  diets  of 
fat  only,  but  in  one  case  less  than  with  carbohydrate  only.  Yeast  was 
fed  with  this  protein-free  food  to  the  extent  of  0.1  gram  per  day  but 
the  exceedingly  small  amount  of  protein  therein  evidently  had  little 

TABLE  8 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  carbohydrate  diet — 
2  grams  cornstarch  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

150 

9.5 

3.2 

10,  288,  000 

90 

9,  568,  000 

91 

12 

395 

22.0 

8.5 

11,600,000 

116 

10,  368,  000 

114 

12 

240 

14.0 

6.0 

9,600,000 

91 

9,696,000 

94 

12 

TABLE  9 


Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  protein-free  diet 

— 5  grams  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

prams 

grams 

grams 

per  cmm. 

days 

195 

11.6 

4.47 

8,976,000 

87 

8,496,000 

90 

9 

255 

14.8 

6.0 

10,896,000 

110 

10,  392,  000 

107 

12 

190 

11.45 

5.3 

10,  000,  000 

110 

10,400,000 

110 

12 

or  no  effect  on  the  speed  of  blood-cell  formation.  In  all  cases  it  was 
impossible  to  keep  the  animals  eating  this  ration  without  the  addition 
of  the  dietary  essential,  vitamin  B.  This  led  to  work  on  diets  without 
added  yeast,  with  yeast  as  a  sole  source  of  nourishment,  and  with  other 
vitamin-containing  products,  such  as  tomato  juice  and  orange  juice 
alone. 

Standard  diet  without  yeast.     Four  rats  were  supplied  with  an  unre- 
stricted quantity  of  standard  diet  and  an  abundance  of  fresh  water,  but 


250 


ZALIA   JENCKS 


no  yeast,  i.e.,  no  vitamin  B,  was  given  to  them;  they  were  bled  accord- 
ing to  the  regular  procedure  and  their  recovery  was  apparently  normal. 
They  ate  less  and  less  as  time  went  on,  as  has  been  the  case  for  all 
animals  with  lack  of  vitamin  B  (12),  and  they  lost  in  weight.  The 
return  to  normal  blood  values  came  in  from  12  to  13  days;  in  three  of 
the  four  cases  hemoglobin  values  were  low.  This  could  not  be  due  to 
lack  of  iron,  for  some  iron  was  eaten  in  the  salt  mixture,  although  more 
iron  would  have  been  consumed  had  yeast  also  been  added  to  the  diet 
(table  10). 

Yeast  only.     Because  of  these  results  with  protein-free  food  and  with 
other  diets  without  yeast,  a  series  of  hemorrhages  was  made  on  rats 
which  were  fed  nothing  but  yeast  during  the  period  of  recovery.    AC- 
TABLE 10 

Blood  regneration  in  adult  rats  after  a  single  hemorrhage;  on  the  standard  diet, 

without  yeast 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

295 

170 

6.48 

11,648,000 

107 

11,292,000 

96 

13 

315 

180 

6.0 

10,  112,  000 

91 

10,288,000 

94 

12 

355 

200 

6.9 

11,584,000 

114 

12,  100,  000 

104 

12 

270 

156 

5.4 

10,  768,  000 

98 

(Sick) 

» 

10,  068,  000 

85 

13 

cordingly  four  animals  were  selected  which  had  responded  in  a  normal 
manner  to  regeneration  after  experimental  anemia  several  weeks  pre- 
vious to  this  time  and  each  was  again  subjected  to  a  hemorrhage. 
These  rats  were  then  fed  0.5  gram  dried  brewery  yeast  daily.  They 
ate  the  yeast  with  great  avidity  as  soon  as  it  was  offered.  This 
amounted  to  practical  starvation,  and  although  they  lost  body  weight 
the  animals  all  regenerated  their  blood  in  from  6  to  8  days  (table  11). 
In  each  case  the  hemoglobin  values  were  equal  to  or  exceeded  the  origi- 
nal. This  group  of  animals  regenerated  their  blood  in  less  time  than 
the  group  without  food. 

As  a  check  for  these  results  with  yeast  alone  a  group  was  given  0.5 
gram  protein  (casein)  daily  to  see  if  the  small  quantity  of  yeast  protein 
was  the  stimulant  to  forming  new  blood  or  whether  it  was  in  truth  the 


251 


food  accessory  in  the  yeast.  The  time  required  for  these  three  animals 
to  regenerate  their  blood  was  7  to  10  days.  The  hemoglobin  values 
were  again  low  in  two  cases  as  for  the  animals  which  were  fed  no  yeast 
(table  12).  The  casein  contained  no  iron  but  the  rats  were  in  iron-wire 
cages  and  continually  gnawed  at  the  wires. 

,;,;    TABLE  11 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  diet  of  yeast  only — 

0.5  gram  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OP 
HEMORRHAGE 

RED  BLOOD 

CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cent 

days 

292 

16.76 

6.5 

11,  184,  000- 

112 

• 

10,416,000 

112 

8 

320 

18.0 

6.2 

9,  134,  000 

90 

9,  648,  000 

98 

7 

290 

16.7 

7.0 

10,784,000 

96 

10,336,000 

98 

6 

355 

20.0 

7.0 

10,  144,  000 

102 

10,656,000 

104 

7 

TABLE  12 


Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  protein  diet- 

0.5  gram  casein  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

• 

days 

147 

9.34 

3.2 

8,704,000 

102 

9,  036,  000 

98 

7 

150 

9.5 

3.0 

12,  000,  000 

108 

9,924,000* 

104 

7 

157 

10.5 

4.1 

11,  040,  000 

87 

11,  770,  000 

91 

10 

*  R.B.C.  constant  3  days 

Water  soluble  vitamin  (£).  Two  products  other  than  yeast,  rich 
in  this  dietary  essential,  were  used  as  a  sole  nutrient  for  a  group  of  rats 
which  had  experienced  hemorrhages.  Orange  juice,  containing  no 
iron  (by  analysis  and  according  to  Sherman  (13))  and  tomato  juice 
containing  no  iron  (by  analysis  and  according  to  Sherman)  were  fed 
to  two  sets  of  animals.  The  orange  juice  was  prepared  by  straining  a 


252 


ZALIA   JENCKS 


mass  of  crushed  pulp  and  juice  through  four  layers  of  clean  cheesecloth. 
This  resulted  in  a  perfectly  clear  fluid  which  was  measured  out  for 
feeding  by  means  of  a  pipette.  The  tomato  juice  was  the  juice  from 
canned  tomatoes,  from  which  the  pulp  and  seeds  were  separated  by 

TABLE  13 

Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  diet  of  orange  juice 
only — vitamin  B — 5  cc.  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 
CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

250 

13.5 

4.9 

12,  384,  000 

114 

11,548,000 

114 

6 

118 

7.7 

2.8 

12/000,  000 

110 

11,  880,  000 

112 

6 

170 

11.2 

3.2 

9,  776,  000 

102 

10,  608,  000 

112 

6 

275 

16. 

5.3 

10,  528,  000 

107 

10,  608,  000 

102 

6 

TABLE  14 


Blood  regeneration  in  adult  rats  after  a  single  hemorrhage;  on  a  diet  of  tomato  juice 

only — 5  cc.  per  day 


BODY  WEIGHT 

TOTAL  BLOOD 

AMOUNT  OF 
HEMORRHAGE 

RED  BLOOD 

CORPUSCLES 

HEMOGLOBIN 

TIME  FOR 
REGENERA- 
TION 

grams 

grams 

grams 

per  cmm. 

days 

400 

22.3 

5.5 

10,768,000 

104 

11,682,000 

107 

6 

225 

13.0 

4.7 

10,  192,  000 

100 

10,  176,  000 

100 

6 

367 

20.5 

8.7 

10,  808,  000 

100 

10,  880,  000 

96 

8 

305 

17.5 

5.6 

8,  592,  000. 

94 

8,  352,  000 

92 

6 

straining  through  four  layers  of  cheesecloth.  This  was  also  measured 
with  a  pipette.  The  four  rats  on  10  cc.  of  orange  juice  daily,  reached 
their  normal  blood  values  in  6  days  (table  13),  those  on  the  tomato 
juice,  5  cc.  daily,  (they  refused  to  eat  as  much  as  10  cc.  per  day)  recovered 
in  from  6  to  8  days;  again  the  hemoglobin  values  varied  (table  14). 


STUDIES   IN   THE   REGENERATION   OF   BLOOD  253 

SUMMARY   OF   RESULTS 

Hemorrhages  of  approximately  one-third  of  the  estimated  total 
blood  volume  can  be  borne  by  albino  rats  without  interfering  with 
complete  recovery.  When  one-half  or  more  of  the  blood  was  removed 
by  hemorrhage,  death  ensued.  The  criteria  used  in  judging  blood 
regeneration  in  the  rat  were  the  return  of  the  number  of  erythrocytes 
and  the  percentage  of  hemoglobin  to  the  values  noted  before  the  animal 
had  sustained  any  loss  of  blood. 

By  varying  the  diet  from  elimination  of  one  factor  to  elimination  of 
all  but  one  factor  therein,  the  speed  of  regeneration  of  blood  was 
changed,  but  with  each  diet  the  organism  replenished  its  normal  supply 
of  blood  to  the  original  values  for  red  cells  and  hemoglobin. 

Protein  permitted  more  rapid  blood  regeneration  than  either  carbo- 
hydrate or  fat  when  fed  as  a  sole  nutrient  (2.0  grams  daily) . 

The  diets  of  vitamin-rich  food,  even  in  very  small  amounts,  gave 
somewhat  more  speedy  regeneration  than  any  other  diet  containing 
one  food  factor  only. 

This  study  of  blood  regeneration  has  not  covered  the  effect  of  iron  on 
blood  formation.  Work  on  this  phase  of  the  problem  is  now  being 
carried  out. 

I  desire  to  express  my  sincere  thanks  to  Prof.  Lafayette  B.  Mendel 
who  outlined  this  problem,  for  his  suggestions  and  interest  throughout 
the  investigation. 

BIBLIOGRAPHY 

(1)  FERRY:  Journ.  Lab.  Clin.  Med.,  1920,  v,  735. 

(2)  OSBORNE  AND  MENDEL:  Journ.  Biol.  Chem.,  1912,  xiii,  233. 

(3)  OSBORNE  AND  MENDEL:  Journ.  Biol.  Chem.,  1913,  xv,  311. 

(4)  OSBORNE  AND  MENDEL:  Journ.  Biol.  Chem.,  1915,  xx,  351. 

(5)  DONALDSON:  The  rat,  Philadelphia,  1915. 

(6)  COHEN  AND  SMITH:  Journ.  Biol.  Chem.,  1919,  xxxix,  489. 

(7)  LAMSON:  Journ.  Pharm.  Exper.  Therap.,  1915,  vii,  169. 

(8)  GEILING  AND  GREEN:  Proc.  Soc.  Exper.  Biol.  and  Med.,  1912,  xviii,  191. 

(9)  CAJORI:  Journ.  Biol.  Chem.,  1920,  xliii,  583. 

(10)  OSBORNE  AND  WAKEMAN:  Journ.  Biol.  Chem.,  1919,  xl,  383. 

(11)  HOOPER  AND  WHIFFLE:  This  Journal,  1918,  xlv,  573. 

(12)  KARR:  Proc.  Soc.  Exper.  Biol.  and  Med.,  1920,  xvii,  84. 

(13)  SHERMAN:  Chemistry  of  food  and  nutrition,  New  York,  1919,  2nd  ed.,  335. 


